1. Field of the Invention
The present invention relates to multivalent vaccine formulations effective against Staphylococcus aureus, including both biofilm and planktonic types of bacterial infections, and to methods of using the formulations in the treatment and prevention of S. aureus infections in subjects.
2. Related Art
One of the most common and costly problems for the U.S. healthcare system is nosocomial infections (26), with S. aureus being the second-leading cause of such infections (4). Methicillin-resistant S. aureus (MRSA) is responsible for 40-60% of all nosocomially-acquired S. aureus infections, and these resistant strains are now considered to be endemic in the hospital setting (36). Community-associated S. aureus strains may also acquire methicillin-resistance (CA-MRSA) and the modern emergence of such strains is of great concern (24, 31, 64).
Recent studies indicate that S. aureus is also the major mediator of prosthetic implant infection (1, 54). The increasing involvement of S. aureus in foreign body-related infections, the rapid development of resistance to multiple antibiotics by these organisms, and the propensity of these infections to change from an acute infection to one that is persistent, chronic and recurrent have led to this organism once again receiving significant attention.
Treating prosthetic implant infections is a complicated process, and a number of staphylococcal defense mechanisms may be responsible for this difficulty as well as the capacity of S. aureus to evade clearance by the host immune response. One of the most important mechanisms utilized by S. aureus to thwart the host immune response and develop into a persistent infection is through the formation of a highly-developed biofilm. A biofilm is defined as a microbe-derived community in which bacterial cells are attached to a hydrated surface and embedded in a polysaccharide matrix (13). Bacteria in a biofilm exhibit an altered phenotype in their growth, gene expression, and protein production (17), and prosthetic medical devices are often a site of chronic infection, because they present a suitable substrate for bacterial adherence, colonization, and biofilm formation. Biofilm formation by S. aureus during prosthetic implant infection makes eradication of this bacteria extremely difficult, due in part to the dramatically increased resistance of bacteria in a biofilm to host defenses (21) and to antibiotics (46, 51), compared to their planktonic counterparts.
Previous vaccine studies have evaluated the efficacy of bacterial polysaccharides, e.g. polysaccharide capsules, exopolysaccharide, and peptidoglycan (10, 20, 38, 41), as well as recombinant protein subunit vaccines (2, 8, 9, 27, 29, 30, 33, 57, 65) against S. aureus infection, but none have demonstrated complete eradication of S. aureus in experimental animal models (2, 8, 9, 27, 29, 30, 33, 57, 65) or passed the rigors of phase III clinical testing (56, 59). Most vaccines evaluated to date do not account for biological redundancy of S. aureus virulence factors, differential protein expression during different modes of growth (exponential growth versus stationary) or type of infection (planktonic versus biofilm), and the lack of antigen conservation amongst relevant clinical isolates. Indeed, a polysaccharide vaccine (StaphVAX) developed using the S. aureus capsular polysaccharide 5 (CP5) and capsular polysaccharide 8 (CP8) conjugated to the Pseudomonas aeruginosa exotoxoid A failed to provide protection in phase III clinical trials against S. aureus-mediated bacteremia in two different cohorts of 1804 and 3600 hemodialysis patients (59). Factors contributing to this failure are the existence of non-encapsulated strains (CP5 and CP8 strains account for 75-80% of isolates) (12) and differential expression as extrapolated from in vitro data indicating that capsular polysaccharide expression is limited to the stationary mode of growth and the absence of CP5 expression in S. aureus bound to endothelial cells (48). The efficacy of the StaphVAX vaccine would, therefore, be limited to planktonic-type infections and ineffective at targeting the humoral response to a S. aureus biofilm.
Similar to the findings with the CP5/CP8 vaccine (20), subunit vaccines developed against the clumping factor A (C1fA) (2, 27), clumping factor B (C1fB) (57), fibronectin binding protein (FnBP) (65), α-Hemolysin (9, 29), Panton-Valentine leukocidin (PVL) (8), and the iron-regulated surface determinant B (IsdB) (30, 33) mediate partial protection in experimental animal models. These subunit vaccines did not provide complete protection, despite the candidate proteins being highly immunogenic in vivo (25, 33, 57) and the resultant antibodies promoting opsonic killing of S. aureus (65). One deficiency of these approaches was relying on a monovalent vaccine to promote protection against the pathogen. S. aureus has nearly 70 virulence factors and functional redundancy amongst these factors may abrogate the effect of neutralizing one factor. Arguably, S. aureus expresses multiple iron acquisition systems: siderophores staphyloferrin A and B transport transferrin to receptors HtsA and SirA (14, 43), an ABC transporter Fhu imports Fe3+ hydroxamates (58), and iron-regulated surface determinant (Isd) B and IsdH receptors that bind hemoglobin/haptoglobin complexes (18, 62), therefore the overall effectiveness of anti-IsdB antibodies that block IsdB-mediated hemoglobin binding may be only a modest effect on iron uptake and the organism's pathogenicity (30). The validity of this argument is exemplified by the cessation of phase III clinical trials of Merck's IsdB vaccine (V710) that failed to provide complete protection (16), despite promising immunogenicity and opsonic killing data from phase II trials (25, 52).
Efficacy of a monovalent vaccine can also be compromised by differential expression of the targeted protein during the course of infection. While S. aureus initiates colonization by binding host extracellular ligands using its adhesin proteins called microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), including the fibronectin-binding protein (FnBP), these factors are mostly down-regulated as the sessile bacteria encapsulate themselves in an extracellular polysaccharide matrix, or biofilm (44, 55). Hence, vaccines designed to target a MSCRAMM will be ineffective at clearance after the bacteria transition into the biofilm phenotype. Evaluation of the MSCRAMM FnBP vaccine demonstrated it provided partial protection against S. aureus in a murine model of sepsis, but the study failed to enumerate bacteria in the blood and/or kidneys to verify bacterial clearance. It is feasible that S. aureus can subvert the humoral response to FnBP, form a sessile biofilm and down-regulate FnBP, and become completely recalcitrant the host response.
A vaccine strategy that circumvents the incomplete protection of monovalent vaccines caused by protein redundancy, differential protein expression, or isolate-specific genetic divergence is the generation of a multifactorial assault using a multivalent subunit vaccine. Stranger-Jones et al. demonstrated a quadrivalent vaccine comprised of surface-exposed proteins: iron-regulated surface determinant A (IsdA), IsdB, and serine aspartate repeat protein D (SdrD), and SdrE increased survival rates against S. aureus-mediated lethal challenge compared to protection afforded by each monovalent variant (61). Although the authors stressed the survival rates after lethal challenge, they omitted enumeration of S. aureus in the kidneys and survival rates beyond 7 days post-infection from the data analysis. These omissions preclude a conclusion to be reached on the vaccine's ability to promote complete bacterial clearance and prevent future complications due to S. aureus persistence via biofilm formation. Overall, the multivalent vaccine had limited efficacy, providing complete protection against only two of five clinical S. aureus isolates tested (61). In addition, comparative analysis of multiple S. aureus genomes found a lack of conservation amongst some surface proteins, including SdrD and SdrE (39), which indicates the limited efficacy of the IsdA/IsdB/SdrD/SdrE vaccine formulation may extend beyond the clinical isolates tested by Stranger-Jones.
Vaccine studies have predominately focused on protection against planktonic-mediated infection by examining sepsis (20, 27, 33, 38, 41, 61, 65) or pneumonia (9), while few studies have incidentally evaluated protection, mediated by popular vaccine candidates, against biofilm infection with experimental endocarditis (2), skin (8, 22, 29), or abscess models (20, 61). As a departure from previous S. aureus vaccine strategies, Brady et al. focused on identifying biofilm upregulated proteins that are immunogenic (4) and established that a multivalent biofilm-based vaccine when coupled with vancomycin treatment could eradicate a biofilm infection, which is traditionally recalcitrant to clearance by either antibiotic treatment or immune response (5). Previous attempts to target the biofilm phenotype, most notably against the staphylococcal intercellular adhesion (PIA) composed of poly-N-acetyl-β-1,6-glucosamine (PNAG) (38, 40, 41), were directed towards the biofilm matrix encapsulating the bacteria versus cell wall-associated proteins. The polysaccharide PNAG vaccine elicited a response that reduced bacterial counts (40), but polysaccharides tend to be weak immunogens and induce antibodies with low opsonic killing activity. In addition, PNAG molecules tend to be loosely associated with the bacterial surface and the acetylated PNAG form is released into suspension (11). Efforts to improve efficacy of the PNAG vaccine have evaluated the deacetylated form of PNAG (dPNAG), which may be retained on the cell surface, conjugated to diphtheria toxoid or a synthetic 9-mer of β-(1→6)-D-glucosamine (GlcNH2) conjugated to tetanus toxoid, but partial protection against multiple S. aureus strains was observed despite improved immunogenicity (22, 38). PIA is generated by enzymes encoded on the icaABDC locus (28), but the presence of the icaABDC locus does not directly correlate to biofilm formation in vitro (32) and the icaABDC locus in S. aureus was dispensable in a subset of in vivo orthopedic prosthesis-associated and catheter-associated infections, which are identified as biofilm-mediated infections (53). While the efficacy of the PNAG vaccine against S. aureus biofilms requires further evaluation, the dispensability of the icaABDC locus in some S. aureus strains isolated from clinical infections suggests that the PNAG vaccine would provide limited protection against S. aureus biofilm infections.
Another consideration for vaccine development is the type of response elicited by the host immune system and the ability of the pathogen to subvert immune mediators using immunoavoidance factors, which may have varied outcomes depending on the host environment. The immune response elicited in vitro against S. aureus or its virulence factors, specifically staphylococcal enterotoxin A or B and the alpha toxin, is a pro-inflammatory Th1-response (3, 7, 15, 42). Indeed, comparison of S. aureus bacteremia outcomes in mice with different genetic backgrounds found that Th1-biased C57BL/6J mice were resistant and Th2-biased BALB/c mice were susceptible to this acute form of S. aureus infection (63). In contrast, a robust Th-1 response was elicited against a S. aureus implant infection in C57BL/6J mice, but the mice were susceptible and developed a chronic infection with 107 CFU/tibia at 49 days post-infection (45). The S. aureus biofilm appears to be recalcitrant to the pro-inflammatory response, which damages host tissue at the infection site generating devitalized sites for S. aureus to colonize. Subsequent evaluation found that Th-2 biased BALB/c mice were resistant to the S. aureus implant infection, and ablation of interleukin-4 or the depletion of Treg cells abrogated the protection against S. aureus in BALB/c mice (46). Th2-mediated resistance to bacterial infection was also revealed for subcutaneous infections with S. aureus, where higher bacterial loads were observed in C57BL/6J mice versus BALB/c mice (45). Increased CXCL-2 expression in the C57BL/6J mice correlated with the susceptibility to subcutaneous infection (45), and may halt the killing activity of polymorphonuclear neutrophils (PMNs) after influx and internalization of S. aureus (23). This differential immune response against S. aureus, which was observed with chronic infections (implant or subcutaneous) versus acute (sepsis), indicates that the choice of mouse strain may impact the outcome of vaccine studies. Most vaccine studies have examined protection against S. aureus using experimental models developed in BALB/c mice (2, 8, 33, 61, 65), while few studies have evaluated vaccine efficacy in C57BL/6J mice (9, 29). Emphasis on BALB/c experimental models to evaluate S. aureus vaccines may yield insight about efficacy against acute or planktonic infections, but these models will be poor evaluators of chronic, biofilm infections and do not represent the immune response bias in humans.
Additional vaccine formulations would add to the arsenal of means used to treat and/or prevent S. aureus infections.